Method for enhancing hematopoiesis with acyl deoxyribonucleosides

ABSTRACT

The invention relates to compositions comprising acyl derivatives of 2&#39;-deoxyribonucleosides. The invention also relates to methods of treating or preventing radiation, mutagen and sunlight-induced biological damage, and methods for improving wound healing and tissue repair, comprising administering the compositions of the present invention to an animal.

This is a Divisional of application Ser. No. 08/309,572, filed Sep. 21,1994, which is a Continuation of Ser. No. 08/149,469, filed Nov. 9,1993, now abandoned, which is a Divisional of Ser. No. 07/487,984, filedFeb. 5, 1990, abandoned, which is a CIP of Ser. No. 07/115,923, filedOct. 28, 1987, now abandoned.

FIELD OF THE INVENTION

This invention relates generally to acyl derivatives ofdeoxyribonucleosides and to the use of those derivatives to deliverexogenous deoxyribonucleosides to animal tissue. More specifically, thisinvention relates to the acyl derivatives of 2'-deoxyadenosine,2'-deoxyguanosine, 2'-deoxycytidine and 2'-deoxythymidine and the use ofthose novel derivatives to deliver the deoxyribonucleosides to animaltissue and thereby to support cellular metabolic functions. Even morespecifically, this invention relates to the use of the novel acylderivatives to treat or prevent a variety of physiological andpathological conditions in cell tissue, including damage by radiation,sunlight, mutagens, wounds, and other conditions.

BACKGROUND OF THE INVENTION

There are fundamentally two possible chemical or biochemical approachesto attenuating the deleterious effects of ionizing radiation onorganisms:

(1) attenuation of initial damage to biological structures, and

(2) improvement or acceleration of recovery.

A number of compounds are known that provide some protection fromionizing radiation when they are present in the body during irradiation.Such compounds are typically antioxidants or free-radical scavengersthat inactivate reactive chemical species formed during irradiationbefore they can damage important biological structures. Prominentexamples of radioprotective compounds include cysteamine,2-beta-amino-ethyl-isothiouronium-Br-HBr (AET), andS-2-(3-aminopropylamino)ethyl phosphorothioic acid (WR-2721). Sincethese compounds must be introduced into the organism before or duringirradiation, they are obviously not useful in situations of unexpectedor accidental exposure. Moreover, these compounds are toxic in humans.

The main possibilities for effective chemical therapy in organisms inwhich irradiation has already occurred are:

(1) to promote repair and recovery of individual cells within theorganism, or

(2) to accelerate or enhance proliferation and/or differentiation ofsurviving stem cells.

Bone marrow and intestinal epithelium are among the tissues mostsensitive to radiation damage; attempts to promote recovery fromirradiation need to focus on the stem cells in these tissues.

There exist several agents which can improve the survival of irradiatedmammals when administered after irradiation. These include theyeast-derived polysaccharide Glucan, and polypeptide cytokines such asInterleukin-1, Granulocyte-Colony Stimulating Factor, andGranulocyte/Macrophage-Colony Stimulating Factor; all of these agentsimprove bone-marrow stem cell proliferation or differentiation. However,their efficacy is modest, producing Dose Reduction Factors less than 1.1when administered after irradiation has already occurred, and their useis complicated by side effects. Moreover, they are all macromoleculeswhich can only be administered parenterally.

There exists a need for compounds which effectively promote recoverywhen administered after exposure to ionizing radiation and which haveimportant pharmaceutical qualities such as nontoxicity and activityafter oral administration. Such agents would be useful in the cases ofaccidental exposure to ionizing radiation, and also in conjunction withradiation therapy for cancer, in order to promote recovery of normaltissue from irradiation. Such agents may also improve recovery fromcertain forms of chemical damage, e.g., bone-marrow suppressionfollowing either accidental or therapeutic exposure to compounds likecyclophosphamide or busulfan, which are both used in cancerchemotherapy.

It has been demonstrated that administration of exogenousdeoxyribonucleic acid (DNA) to experimental animals after exposure toionizing radiation can result in improved survival and functionalrecovery. Kanazir et al., Bull. Inst. Nuc. Sci. "Boris Kidrich"9:145-153 (1959); Wilczok, T., et al., Int. J. Rad. Biol. 9:201-211(1965); Golba, S., et al., Int. J. Rad. Biol. 13:261-268 (1967); U.S.Pat. No. 3,803,116.

Studies in cell cultures in vitro suggest that the actual restorativeagents are deoxyribonucleosides, the enzymatic degradation products ofDNA. Petrovic, D., et al., Int. J. Rad. Biol. 18:243-258 (1970).However, depolymerized DNA or deoxyribonucleosides administered toanimals were ineffective in promoting survival or recovery afterirradiation. Kanazir et al., Bull. Inst. Nuc. Sci. "Boris Kidrich"9:145-153 (1959). There is reason to believe that this apparentcontradiction is due to the rapid catabolism of deoxyribonucleosides invivo by the enzymes in plasma and various organs. Thus, afteradministration of deoxyribonucleosides to rodents, tissues are exposedto effective concentrations for less than five minutes. Beltz et al.,Bioch. Biophys., Acta. 297:258-267 (1973). In cell cultures, optimumsurvival after irradiation was found when deoxyribonucleosides werepresent in the culture medium for at least three hours. When DNA isadministered parenterally, it is probably gradually depolymerized togive a sustained release of free deoxyribonucleosides into thecirculation.

There may be other physiological or pathological conditions of mammaliantissue wherein the supply of exogenous deoxyribonucleosides may havetherapeutic applications. Newman et al., Am. J. Physiol. 164:251-253(1951), disclose a study in rats subjected to partial hepatectomy. Thecourse of liver regeneration was followed for eleven days. The livers ofrats treated with DNA regenerated significantly faster than did liversof untreated animals. It is likely that deoxyribonucleosides were theactual active agents in this study, since DNA is a large molecule thatis not taken up efficiently by mammalian cells. Similarly, DNA appliedto dermal wounds has been found to accelerate some aspects of thehealing process, e.g., formation of granulation tissue. Dumont, Ann.Surg. 150:799-807 (1959); Marshak et al., Proc. Soc. Exp. Biol. Med.58:62-63 (1945); Nicolau et al., Der Hautartzt 17:512-515 (1966). Yaneand Kitano, U.S. Pat. No. 4,656,896, disclose evidence of beneficialeffects of parenterally administered DNA in the treatment of gastriculcers in rats.

In these examples, it is likely that the effect of DNA was related toits gradual degradation, resulting in the release ofdeoxyribonucleosides over a prolonged period. DNA is not, however, asuitable pharmaceutical agent to administer to humans, either orally orparenterally. In the case of oral administration, nucleosides releasedfrom DNA would mainly be degraded by enzymes in the intestinal lumen, inthe intestinal walls, in plasma, and in the liver, rather than beingavailable to tissues. Problems with parenterally administered DNAinclude possible antigenicity (exacerbated by adhering proteins whichare difficult to remove during extraction), nonuniformity betweenbatches, and possible undesirable effects not related to nucleosiderelease, e.g., enhancement of interferon release from lymphocytes, whichis a known effect of double-stranded nucleic acid.

The administration of deoxyribonucleosides has heretofore beencontemplated for the reversal of obvious deficiencies ofdeoxyribonucleotides (e.g., thymidine administration to reverse toxicitycaused by methotrexate, an antineoplastic agent which inhibits thymidinenucleotide biosynthesis; administration of deoxycytidine to reversearabinosyl cytosine toxicity, or in people with deficiencies ofparticular enzymes (e.g., purine nucleoside phosphorylase) thatultimately result in impaired deoxyribonucleotide synthesis). Thymidineadministration has also been considered as an antineoplastic treatment,since, in high concentrations, thymidine has cytostatic or cytotoxicproperties.

However, the invention disclosed herein pertains to the recognition thatunexpected beneficial effects may be obtained after administration ofsupraphysiological quantities of mixtures of deoxyribonucleosides insuch a manner that they are available to tissues for a sustained period;this goal may be best accomplished through the use of thedeoxyribonucleoside derivatives of the invention.

OBJECTS OF THE INVENTION

While the strategy of delivering DNA and/or deoxyribonucleosides tophysiologically or pathologically damaged tissue has been recognized,the art has heretofore failed to provide satisfactory methods forintroducing deoxyribonucleosides in sufficiently high and reliableamounts in vivo to successfully treat the pathological and physiologicalconditions and to promote cellular repair and survival of the animal.Moreover, although a variety of compounds have been developed whichprotect animals against some effects of ionizing radiation or chemicalmutagens, deoxyribonucleosides provided to tissues for a sufficient timehave the greatest clinical potential for post-exposure treatment of suchdamage. Clinical implementation of this strategy, however, awaitsdevelopment of satisfactory and convenient methods for deliveringadequate quantities of deoxyribunucleosides to tissues in vivo.Similarly, full appreciation and clinical implementation of the capacityof deoxyribonucleosides to promote wound healing or tissue repair awaitsdevelopment of satisfactory methods for their delivery to tissues invivo.

It is thus a primary object of this invention to identifypharmaceutically acceptable compounds which can efficiently be used todeliver pharmacologically effective amounts of deoxyribonucleosides ortheir respective derivatives to animal tissue.

It is still a further object of this invention to provide a family ofdeoxyribonucleoside derivatives which can be effectively administeredorally or parenterally, which have minimal toxicity, and which can beadministered to animals and humans to effectively promote cellularrepair in a number of physiological and pathological conditions and topromote survival of the animal when administered after exposure toradiation has occurred.

It is still a further and related object of this invention to providecertain derivatives of 2'-deoxyadenosine, 2'-deoxyguanosine,2'-deoxycytidine, and 2'-deoxythymidine which, when administered to ananimal, will deliver those deoxyribonucleosides to the animal tissue.

It is a related object of this invention to substantially improve thebioavailability of 2'-deoxyadenosine, 2'-deoxyguanosine,2'-deoxycytidine, and 2'-deoxythymidine by enhancing the transport ofthese deoxyribonucleosides across the gastrointestinal tract and otherbiological membranes.

It is still a further and more specific object of this invention toprovide a family of deoxyribonucleoside derivatives for the treatment ofa variety of liver, bone, skin, hematological, and other pathologicaland physiological conditions.

It is still a further object of this invention to providedeoxyribonucleoside derivatives and methods for using those derivativeswhich are safe, inexpensive, and which accelerate the normal cellularprocesses of regeneration and healing.

SUMMARY OF THE INVENTION

These and other objects of the invention are achieved by theadministration of certain acyl derivatives of 2'-deoxyadenosine,2'-deoxyguanosine, 2'-deoxycytidine, and 2'-deoxythymidine. These acylderivatives can be used to prevent or treat radiation, sunlight andmutagen-induced cellular damage, to improve the healing of wounds, orrepair damaged tissues, and in the treatment of other physiological andpathological tissue conditions.

While the prior art discloses some acylated derivatives ofdeoxyribonucleosides, their substituents (e.g., pivaloate, isobutyrate,benzoate, or adamantoate) were selected for properties related toutility as protecting groups in chemical synthesis (e.g., ofoligonucleotides), and are not generally acceptable for administrationto animals. The novel compounds disclosed herein are preferred becauseof their nontoxic substituents. These present minimal hazard to theorganism to which they are administered and can be selected to yielddesirable pharmaceutical and pharmacological properties without undueexperimentation.

Acylated derivatives of some antineoplastic and antiviral nucleosideanalogs have been utilized as prodrugs of these cytotoxic agents.However, very different biochemical and physiological issues areinvolved in improving the therapeutic index of toxic nucleoside analogsversus the delivery of the nontoxic deoxyribonucleosides in appropriatequantities and combinations for improving tissue repair or regeneration,as in the present invention.

A major aspect of the invention is the recognition that acyl derivativesof deoxyribonucleosides, particularly when derivatives of two or moredeoxyribonucleosides are combined, have unexpected therapeuticproperties. This is evidenced in the data concerning survival ofirradiated mice. The invention also includes novel classes ofderivatives that are particularly desirable in terms of both efficacyand safety.

Broadly, the acyl derivatives of 2'-deoxyadenosine are those having theformula (I) ##STR1## wherein R is hydrogen or an acyl radical of ametabolite other than acetyl, with the proviso that at least one R isnot hydrogen, or a pharmaceutically acceptable salt thereof.

The preferred acyl derivatives of 2'-deoxyadenosine are those having theformula (I) ##STR2## wherein R is H or an acyl group derived from acarboxylic acid selected from one or more of the group consisting ofpyruvic acid, lactic acid, enolpyruvic acid, an amino acid, a fatty acidother than acetic acid, lipoic acid, nicotinic acid, pantothenic acid,succinic acid, fumaric acid, p-aminobenzoic acid, betahydroxybutyricacid, orotic acid, and carnitine, with the proviso that at least one Ris not hydrogen, or a pharmaceutically acceptable salt thereof.

Broadly, the acyl derivatives of 2'-deoxyguanosine are those having theformula (II) ##STR3## wherein R is hydrogen or an acyl radical of ametabolite other than acetyl, with the proviso that at least one R isnot hydrogen, or a pharmaceutically acceptable salt thereof.

The preferred acyl derivatives of 2'-deoxyguanosine are those having theformula (II) ##STR4## wherein R is H or an acyl group derived from acarboxylic acid selected from one or more of the group consisting ofpyruvic acid, lactic acid, enolpyruvic acid, an amino acid, a fatty acidother than acetic acid, lipoic acid, nicotinic acid, pantothenic acid,succinic acid, fumaric acid, p-aminobenzoic acid, betahydroxybutyricacid, orotic acid, and carnitine, with the proviso that at least one Ris not hydrogen, or a pharmaceutically acceptable salt thereof.

Broadly, the acyl derivatives of 2'-deoxycytidine are those having theformula (III) ##STR5## wherein R is hydrogen or an acyl radical of ametabolite other than acetyl, with the proviso that at least one R isnot hydrogen, or a pharmaceutically acceptable salt thereof.

The preferred acyl derivatives of 2'-deoxycytidine are those having theformula (III) ##STR6## wherein R is H or an acyl group derived from acarboxylic acid selected from one or more of the group consisting ofpyruvic acid, lactic acid, enolpyruvic acid, an amino acid, a fatty acidother than acetic acid, lipoic acid, nicotinic acid, pantothenic acid,succinic acid, fumaric acid, p-aminobenzoic acid, betahydroxybutyricacid, orotic acid, and carnitine, with the proviso that at least one Ris not hydrogen, or a pharmaceutically acceptable salt thereof.

Broadly, the acyl derivatives of 2'-deoxythymidine are those having theformula (IV) ##STR7## wherein R is hydrogen or an acyl radical of ametabolite other than a fatty acid having less than five carbon atoms,with the proviso that at least one R is not hydrogen, or apharmaceutically acceptable salt thereof.

The preferred acyl derivatives of 2'-deoxythymidine are those having theformula (IV) ##STR8## wherein R is H or an acyl group derived from acarboxylic acid selected from one or more of the group consisting ofpyruvic acid, lactic acid, enolpyruvic acid, an amino acid, a fatty acidcontaining 5 or more carbon atoms, lipoic acid, nicotinic acid,pantothenic acid, succinic acid, fumaric acid, p-aminobenzoic acid,betahydroxybutyric acid, orotic acid and carnitine, with the provisothat at least one R substituent is not hydrogen, or a pharmaceuticallyacceptable salt thereof.

The acyl derivatives of 2'-deoxythymidine may also be those having theformula (V) ##STR9## wherein R" is hydrogen or an acyl radical of ametabolite, with the proviso that the R" on nitrogen is not hydrogen, ora pharmaceutically acceptable salt thereof.

Preferred acyl derivatives of 2'-deoxythymidine are those having theformula (V) ##STR10## wherein R" is H or an acyl group derived from acarboxylic acid selected from one or more of the group consisting ofpyruvic acid, lactic acid, enolpyruvic acid, an amino acid, a fattyacid, lipoic acid, nicotinic acid, pantothenic acid, succinic acid,fumaric acid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid,and carnitine, with the proviso that the R" on nitrogen is not hydrogen,or a pharmaceutically acceptable salt thereof.

The invention also includes compounds having formulae I-IV wherein theribose moiety is monoacylated at the 3' or 5' position with thederivative of a fatty acid and includes 3',5'-diacylated derivatives ofcompounds I-IV wherein at least one such substituent is derived from afatty acid having 5 or more carbon atoms.

The acyl derivatives of 2'-deoxyadenosine, 2'-deoxyguanosine,2'-deoxycytidine, and 2'-deoxythymidine having formulae I, II, III, andV, desirably are substituted with an acyl derivative of a carboxylicacid having 3-22 carbon atoms.

Where acyl derivatives of any of the compounds of formulae I-V aresubstituted by an acyl group derived from an amino acid, the amino acidis desirably selected from the group consisting of glycine, the L formsof alanine, valine, leucine, isoleucine, phenylalanine, tyrosine,proline, hydroxyproline, serine, threonine, cysteine, cystine,methionine, tryptophan, aspartic acid, glutamic acid, arginine, lysine,histidine, ornithine, and hydroxylysine.

In a preferred embodiment of the invention, a mixture of at least twoacyl derivatives of 2'-deoxyadenosine, 2'-deoxyguanosine,2'-deoxycytidine, and 2'-deoxythymidine is used. Said compositionscontain an effective amount of each of at least two compounds selectedfrom at least two of the groups of compounds having the formulae##STR11## wherein R₁, R₂, and R₃ are the same or different and each is Hor an acyl group derived from a carboxylic acid, provided that at leastone of said substituents R₁, R₂, and R₃ in each of said groups ofcompounds is not hydrogen, or pharmaceutically acceptable salts thereof.In a preferred embodiment, R₁, R₂, and R₃ are the same or different andeach is B or an acyl group derived from a carboxylic acid selected fromthe group consisting of an amino acid, an unbranched fatty acidcontaining 2 to 22 carbon atoms, a dicarboxylic acid containing 3 to 22carbon atoms, and an optionally substituted benzoyl or heterocyclicaromatic carboxylic acid that is substantially nontoxic. Preferredoptionally substituted benzoyl or heterocyclic carboxylic acids includenicotinic acid, and p-aminobenzoic acid.

In another preferred embodiment of the invention, a compositioncomprising a mixture of an effective amount of at least three compoundsselected from at least three of the groups of compounds having theformulae I-IV, shown above, is used. In still another preferredembodiment, a composition comprising a mixture of an effective amount ofat least four compounds selected from at least four of the groups ofcompounds having the formulae I-IV, shown above, is used.

Further substantial benefits may be obtained, particularly where thecompositions of the invention are used to ameliorate the effects ofradiation, if a radioprotective compound is included together with oneor more of the acyl deoxyribonucleosides. The radioprotective compoundsmay be those selected from the group consisting of WR-2721, NAC, DDC,cysteamine, 2-mercaptoethanol, mercaptoethylamine dithiothreitol,glutathione, 2-mercaptoethanesulfonic acid, WR-1065, nicotinamide,5-hydroxytryptamine, 2-beta-aminoethyl-isothiouronium-Br-Hbr, glucans,GLP/B04, GLP/B05, OK-432, Biostim, PSK, Lentinan, Schizophyllan,Rhodexman, Levan, Mannozym, MVE-2, MNR, MMZ, IL-1, TNF, thymic factorTF-5, glutathione peroxidase, superoxide dismutase, catalase,glutathione reductase, glutathione transferase, selenium, CdCl₂, MnCl₂,Zn acetate, Vitamin A, beta carotene, prostaglandins, tocopherol,methylene blue and PABA.

The invention is also embodied in pharmaceutical compositions whichcomprise one or more of the novel deoxyribonucleosides together with apharmaceutically acceptable carrier. In addition, known acetylderivatives of the 2'-deoxyadenosine, 2'-deoxyguanosine,2'-deoxycytidine and 2'-deoxythymidine as well as the fatty acidderivatives of thymidine wherein the acyl group contains 3 or 4 carbonatoms may be used alone, in combination with one another or incombination with one or more novel compounds, in pharmaceuticalcompositions of the invention. The composition may further include aradioprotective compound as described. The compositions may be in theform of a liquid, a suspension, a tablet, a dragee, an injectablesolution, a topical solution, or a suppository.

A skin lotion may be advantageously prepared by combining an effectiveamount of one or more of the acyl deoxyribonucleosides of the inventiontogether with a suitable carrier. Such a skin lotion advantageouslycontains from 0.1 to 5 percent by weight of the deoxyribonucleosidesand, if desirable, the radioprotective compound.

The pharmaceutical compositions of the invention can also be embodied inbioerodible microcapsules, the microcapsules desirably being selectedfrom the group consisting of polylactate or lactate-glycolatecopolymers.

It is believed that the delivery of exogenous deoxyribonucleosides tothe tissue of an animal can be effectively achieved by administering tothat animal an effective amount of an acyl derivative of adeoxyribonucleoside of formulae I-V. By enhancing the delivery ofexogenous deoxyribonucleosides, and thereby increasing theirbioavailability, it may be possible to treat physiological orpathological conditions of the tissues of an animal by essentiallysupporting some metabolic functions thereof. Without being bound bytheory, the invention may work, as well, by increasing thebioavailability of nucleoside anabolites, e.g., nucleotides ornucleotide-derived cofactors. Administration of the nucleosides per seincreases their bioavailability but, due to rapid extracellularcatabolism, this may not result in sustained elevation of cellularnucleotide levels. At lower nucleoside levels there is rapid uptake andutilization by the cells whereas at higher levels there is saturationand the excess is degraded. The invention is believed to work bydelivering a sustained supply of nucleoside at lower levels.

The specific conditions where advantages may be achieved using thecompounds, compositions, and methods of the invention include situationswhere improvement of DNA repair or improvement of stem celldifferentiation and proliferation are useful. Such conditionsparticularly include: (1) treating or preventing damage due to ionizingor ultraviolet irradiation; (2) improving restoration of hematopoiesisin the case of diminished bone marrow function due to ionizingradiation, chemical damage (e.g., side effects of anticancer orantiviral treatments), or disease; and (3) accelerating regeneration andrepair of various damaged tissues, e.g., in healing of wounds and burns,or in promoting regeneration of damaged liver tissue. In treating all ofthese conditions, a compound of the invention, with or withoutadditional carriers, radioprotective compounds, and other adjuvants, isadministered to an animal, in particular, a human.

Administration of the acylated derivatives offers certain advantagesover the nonderivatized compounds. The acyl substituents can be selectedto increase the lipophilicity of the nucleoside, thus improving itstransport from the gastrointestinal tract into the bloodstream. Theacylated derivatives are effective when administered orally and may beapplied topically in some situations. The acylated derivatives areresistant to catabolism by nucleoside deaminases and nucleosidephosphorylases in the intestine, liver, other organs, and thebloodstream. Thus, administration of the acylated derivatives of theinvention, either orally, parenterally, or topically, allows sustaineddelivery of desirable combinations and quantities ofdeoxyribonucleosides to the tissues of an animal, since the acylsubstituents are gradually removed by enzymes (esterases and peptidases)in plasma and tissues, releasing free deoxyribonucleosides over time.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the rates of degradation ofdeoxyribonucleosides in plasma. The following abbreviations are used:

dT=2'-deoxythymidine

dC=2'-deoxycytidine

dG=2'-deoxyguanosine

dA=2'-deoxyadenosine.

FIG. 2 is a graph illustrating the rapid catabolism of deoxyadenosine,and the gradual deacylation of adenosine derivatives (to yielddeoxyadenosine) in plasma.

FIG. 3 is a graph illustrating the rapid catabolism of deoxyadenosinederivatives (to yield deoxyadenosine) in liver extract.

FIG. 4 is a graph illustrating plasma thymidine concentrations afteroral administration of thymidine or di-O-acetylthymidine to rats.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A "metabolite" is a chemical compound that is formed by, or participatesin, a metabolic reaction. In the context of this application,metabolites include not only carboxylic acids known to be synthesizedwithin the human body, but also naturally occurring (but perhapssynthesized rather than extracted) carboxylic acids that might bederived from other animal or plant sources. The limiting criteria arethat the compound should be substantially nontoxic and biocompatible,and should readily enter into metabolic pathways in vivo, so as topresent essentially no toxicity during long-term consumption in thedoses proposed. It is preferable that the compounds be metabolizedrather than excreted intact (or conjugated through detoxificationreactions), as concentration of carboxylic acids within the kidney maylead to undesirable excessive acidity. Therefore, carboxylic acids thatnormally or easily participate in intermediary, catabolic, or anabolicmetabolism are preferred substituents.

The term "pharmaceutically acceptable salts" means salts withpharmaceutically acceptable acid addition salts of thedeoxyribonucleoside derivatives, which include, but are not limited to,sulfuric, hydrochloric, or phosphoric acids.

The term "coadministered" means that at least two of the acylatedderivatives of the invention are administered during a time framewherein the respective periods of pharmacological activity overlap.

"Acyl derivatives" means derivatives of a 2'-deoxyribonucleoside inwhich a substantially nontoxic organic acyl substituent derived from acarboxylic acid is attached to one or more of the free hydroxyl groupsof the ribose moiety of the deoxyribonucleoside with an ester linkageand/or where such a substituent is attached to a primary or secondaryamine in the pyrimidine ring of deoxycytidine or deoxythymidine, or inthe purine ring of deoxyadenosine or deoxyguanosine, with an amidelinkage. Such acyl substituents are derived from carboxylic acids whichinclude, but are not limited to, compounds from the group consisting oflactic acid, an amino acid, a fatty acid, nicotinic acid, dicarboxylicacids, p-aminobenzoic acid, and orotic acid. Preferred acyl substituentsare compounds which are normally present in the body, either as dietaryconstituents or as intermediary metabolites, which are essentiallynontoxic when cleaved from the deoxyribonucleoside in vivo.

"Amino acids" include, but are not limited to, glycine, the L forms ofalanine, valine, leucine, isoleucine, phenylalanine, tyrosine, proline,hydroxyproline, serine, threonine, cysteine, cystine, methionine,tryptophan, aspartic acid, glutamic acid, arginine, lysine, histidine,ornithine, hydroxylysine, carnitine, and other naturally occurring aminoacids.

"Fatty acids" are aliphatic carboxylic acids having 2-22 carbon atoms.Such fatty acids may be saturated, partially saturated orpolyunsaturated.

"Dicarboxylic acids" are fatty acids with a second carboxylic acidsubstituent.

Preferred acyl derivatives of 2-deoxyribonucleosides for enhancingtransport across biological membranes are those which are morelipophilic than are the parent nucleosides. In general, lipophilic acylnucleoside derivatives have acyl substituents which are nonpolar (asidefrom the carboxylate group). Lipophilic acyl substituents includeespecially groups derived from fatty acids containing 2 to 22 carbonatoms. One of ordinary skill in the art can determine whether aparticular acyl-substituted nucleoside derivative is more lipophilicthan the underivatized nucleoside using standard techniques, i.e.,comparison of the partition coefficients determined in water-octanolmixtures.

Following passage of the acylated nucleoside derivative from thegastrointestinal tract into the bloodstream, or across other biologicalmembranes, the acyl substituents are cleaved by plasma and tissueesterases (or amidases) to give the free nucleosides. The preferred acylgroups of the invention are naturally occurring metabolites in the body,or are compounds which readily enter intermediary metabolic pathways.Thus they offer little toxicity when released in vivo by endogenousesterases or amidases.

It is also possible to prepare acyl nucleoside derivatives which containboth polar and nonpolar acyl substituents. The polar acyl group willretard passage of the nucleoside derivative from the gastrointestinaltract, allowing for a more sustained delivery of the compound into thebloodstream after a single dose. The polar group may be cleaved byesterases, amidases, or peptidases present in the intestinal tract togive a nucleoside with a nonpolar acyl substituent which may thenefficiently enter the circulation. Polar acyl substituents may be chosenby one of ordinary skill in the art, without undue experimentation,which are cleaved at a faster rate than are nonpolar acyl substituents.Preferred such substituents are basic amino acids (lysine or arginine),acidic amino acids (glutamate or aspartate), or dicarboxylic acids.

For parenteral injection, acyl derivatives with polar substituents,which are therefore water soluble yet resistant to premature degradationor elimination, may also be used with advantage.

PREFERRED COMPOUNDS OF THE INVENTION

The preferred compounds of the invention are

(1) acyl derivatives of 2'-deoxyadenosine, having the formula ##STR12##wherein R₁, R₂, and R₃ may be the same or different and each is hydrogenor an acyl group derived from

(a) an unbranched fatty acid with 3 to 22 carbon atoms,

(b) an amino acid selected from the group consisting of glycine, the Lforms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cysteine, aspartic acid, glutamicacid, arginine, lysine, histidine, carnitine, and ornithine,

(c) nicotinic acid, or

(d) a dicarboxylic acid having 3 to 22 carbon atoms, provided that

(i) not all of R₁, R₂, and R₃ are H, and

(ii) where R₃ is not H, then R₁ and/or R₂ may also be acetyl,

or a pharmaceutically acceptable salt thereof;

(2) acyl derivatives of 2'-deoxyguanosine having the formula ##STR13##wherein R₁, R₂, and R₃ may be the same or different and each is hydrogenor an acyl group derived from

(a) an unbranched fatty acid with 3 to 22 carbon atoms,

(b) an amino acid selected from the group consisting of glycine, the Lforms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cysteine, aspartic acid, glutamicacid, arginine, lysine, histidine, carnitine, and ornichine,

(c) nicotinic acid, or

(d) a dicarboxylic acid having 3 to 22 carbon atoms, provided that

(i) not all of R₁, R₂, and R₃ are H, and

(ii) where R₃ is not H, then R₁ and/or R₂ may also be acetyl,

or a pharmaceutically acceptable salt thereof;

(3) acyl derivatives of 2'-deoxycytidine, having the formula ##STR14##wherein R₁, R₂, and R₃ may be the same or different and each is hydrogenor an acyl group derived from

(a) an unbranched fatty acid with 3 to 22 carbon atoms,

(b) an amino acid selected from the group consisting of glycine, the Lforms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cysteine, aspartic acid, glutamicacid, arginine, lysine, histidine, carnitine, and ornithine,

(c) nicotinic acid, or

(d) a dicarboxylic acid having 3 to 22 carbon atoms, provided that

(i) not all of R₁, R₂, and R₃ are H, and

(ii) where R₃ is not H, then R₁ and/or R₂ may also be acetyl,

or a pharmaceutically acceptable salt thereof;

(4) acyl derivatives of 2'-deoxythymidine, having the formula ##STR15##wherein R₁ is an acyl group derived from (a) an unbranched fatty acidwith 3 to 15 or 17 to 22 carbon atoms,

(b) an amino acid selected from the group consisting of glycine, the Lforms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cysteine, aspartic acid, glutamicacid, arginine, lysine, histidine, carnitine, and ornithine,

(c) nicotinic acid, or

(d) a dicarboxylic acid having 3 to 22 carbon atoms, and R₂ and R₃ areH, or a pharmaceutically acceptable salt thereof;

(5) acyl derivatives of 2'-deoxythymidine, having the formula ##STR16##wherein R₁ is H, R₂ is an acyl group derived from (a) an unbranchedfatty acid with 3 to 13 or 15 to 22 carbon atoms,

(b) an amino acid selected from the group consisting of glycine, the Lforms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cysteine, aspartic acid, glutamicacid, arginine, lysine, histidine, and ornithine,

(c) nicotinic acid, or

(d) a dicarboxylic acid with 3 to 22 carbon atoms,

and R₃ is H or a pharmaceutically acceptable salt thereof;

(6) acyl derivatives of 2'-deoxythymidine, having the formula ##STR17##wherein R₁ and R₂ may be the same or different and each is an acyl groupderived from

(a) an unbranched fatty acid with 5 to 22 carbon atoms,

(b) an amino acid selected from the group consisting of glycine, the Lforms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cysteine, aspartic acid, glutamicacid, arginine, lysine, histidine, carnitine, and ornithine,

(c) nicotinic acid, or

(d) a dicarboxylic acid with 3 to 22 carbon atoms,

and R₃ is H, or a pharmaceutically acceptable salt thereof; and

(7) acyl derivatives of 2'-deoxythymidine, having the formula ##STR18##wherein R₁ and R₂ are the same or different and each is an acyl groupderived from

(a) an unbranched fatty acid with 2 to 22 carbon atoms,

(b) an amino acid selected from the group consisting of glycine, the Lforms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cysteine, aspartic acid, glutamicacid, arginine, lysine, histidine, carnitine, and ornithine,

(c) nicotinic acid, or

(d) a dicarboxylic acid with 3 to 22 carbon atoms, and

R₃ is an acyl group derived from an optionally substituted benzoyl orheterocyclic carboxylic acid that is substantially nontoxic, or apharmaceutically acceptable salt thereof.

The preferred acyl derivatives of 2'-deoxyadenosine are those wherein R₁is an acyl group derived from an unbranched fatty acid with 6 to 16carbon atoms, R₂ is H or an acyl group derived from an unbranched fattyacid with 6 to 16 carbon atoms, and R₃ is H or an acyl group derivedfrom an amino acid with an acidic or basic side chain.

The preferred acyl derivatives of 2'-deoxyguanosine are those wherein R₁is an acyl group derived from an unbranched fatty acid with 6 to 16carbon atoms, R₂ is H or an acyl group derived from an unbranched fattyacid with 6 to 16 carbon atoms or an amino acid with an acidic or basicside chain, and R₃ is H or an acyl group derived from an amino acid withan acidic or basic side chain.

The preferred acyl derivatives of 2'-deoxycytidine are those wherein R₁is an acyl group derived from an unbranched fatty acid with 6 to 16carbon atoms, R₂ is H or an acyl group derived from an unbranched fattyacid with 6 to 16 carbon atoms, and R₃ is H or an acyl group derivedfrom an amino acid with an acidic or basic side chain.

The preferred acyl derivatives of 2'-deoxythymidine (4) are thosewherein R₁ is an acyl group derived from an unbranched fatty acid with 6to 15 carbon atoms.

The preferred acyl derivatives of 2'-deoxythymidine (5) are thosewherein R₂ is an acyl group derived from an unbranched fatty acid with16 carbon atoms.

The preferred acyl derivatives of 2'-deoxythymidine (6) are thosewherein R₁ and R₂ are the same or different and each is an acyl groupderived from an unbranched fatty acid with 6 to 16 carbon atoms.

The preferred acyl derivatives of 2'-deoxythymidine (7) are thosewherein R₁ and R₂ are the same or different and each is an acyl groupderived from an unbranched fatty acid with 6 to 16 carbon atoms and R₃is an acyl group derived from nicotinic acid, benzoic acid, orpara-aminobenzoic acid.

Therapeutic Uses

The lipophilic acyl deoxyribonucleoside derivatives of the invention areuseful for enhancing the transport of the deoxyribonucleosides acrossbiological membranes including the gastrointestinal tract in animals andthereby increase the bioavailability of the deoxyribonucleosides.Foremost among such animals are humans; however, the invention is notintended to be so limited, it being within the contemplation of theinvention to treat all animals which may experience a beneficial effectfrom the administration of the acyl deoxyribonucleosides of theinvention.

The compositions of the present invention may be administered to ananimal either before or after exposure to radiation, sunlight ormutagens. The acyl derivative form of the deoxyribonucleosides providesan orally effective means for delivery of deoxyribonucleosides totissues. These derivatives may also be given parenterally or topically.Administration of the derivatives avoids the problem of rapid catabolismby gastrointestinal, liver and plasma enzymes.

As shown in FIG. 1, free deoxyguanosine (dG) and deoxyadenosine (dA) aredegraded in plasma extremely rapidly.

The fates of deoxyadenosine, 5'-O-acetyldeoxyadenosine, and5'-O-valeryldeoxyadenosine in plasma are shown in FIG. 2. Each of thesecompounds was added to separate aliquots of rat plasma, at initialconcentrations of 20 micromolar. The plasma was sampled at various timepoints, and the desired compounds were assayed by liquid chromatography.

Deoxyadenosine (dA) is very rapidly degraded in plasma, disappearingwithin 10 minutes. Administration of this compound to an animal or humansubject would make deoxyadenosine available to tissues for a very shortperiod of time.

5'-O-acetyldeoxyadenosine and 5'-O-valeryldeoxyadenosine are, however,deacylated in plasma (to form deoxyadenosine) over a period of severalhours. Therefore, administration of either of these compounds wouldresult in prolonged availability of deoxyadenosine to tissues.

The fates of deoxyadenosine, 5'-O-acetyldeoxyadenosine, and5'-O-valeryldeoxyadenosine in liver extract are shown in FIG. 3. Each ofthese compounds was added to separate aliquots of an aqueous extract ofrat liver, at initial concentrations of 20 micromolar. The extract wassampled at various time points, and the desired compounds were assayedby liquid chromatography.

Deoxyadenosine (dA) is extremely rapidly degraded in plasma,disappearing within 1 minute. The initial degradation product isdeoxyinosine, which is not directly reutilizable by tissues.Administration of deoxyadenosine per se to an animal or human subjectwould make deoxyadenosine available to tissues for only a very shortperiod of time.

5'-O-acetyldeoxyadenosine and 5'-O-valeryldeoxyadenosine are, however,deacylated in liver extract (to form deoxyadenosine) over a period ofmore than 1 hour. Therefore, administration of either of these compoundswould result in prolonged availability of deoxyadenosine to liver orother organs.

Thus a mixture of several different acyl derivatives of eachdeoxyribonucleoside in an administered dose may be selected to provideoptimal bioavailability. A composition containing3',5'-diacetyl-2'-deoxycytidine, and 5'-palmitoyl-2'-deoxycytidine (andcorresponding derivatives of other deoxyribonucleosides) provides a moreprolonged bioavailability of nucleosides after a single dose than doesadministration of a single acyl derivative of each nucleoside. Thus,after administration of the mixture described above, the acetylatedcompound is relatively rapidly deacetylated, yielding free deoxycytidine(or other desired deoxyribonucleosides) shortly after administration.The 5'-palmitoyl derivative is deacylated more slowly, providingadditional free deoxycytidine after the deoxycytidine derived from 3',5'-diacetyl-2'-deoxycytidine has been metabolized by tissues.

The acyl deoxyribonucleoside composition may be formulated as part of asuntan lotion that may be applied before or after exposure to sunlight.The suntan lotion may also comprise one or more sun blockers such asPABA, esters of PABA, and other non-PABA chemical sunscreens. The acyldeoxynucleotides are absorbed by the skin and taken up by cells. Theacyl deoxyribonucleosides are then cleaved by tissue esterases to givefree deoxyribonucleosides in amounts effective for repair ofsunlight-induced damage. The combination of the acyl deoxyribonucleosidecompositions and a sun blocker such as PABA offers maximal protection ofthe skin from the sun.

The acyl deoxyribonucleoside compositions of the invention also find usein ameliorating some of the effects of aging by providing a high andsustained level of deoxyribonucleosides to enhance the natural DNArepair processes of cells, and thereby, treating the naturally occurringprogressive accumulation of damage to DNA which occurs on aging.Compositions for treatment or amelioration of the effects of aging maybe applied topically, in the form of a skin lotion, or may beadministered orally or parenterally.

There are conditions other than radiation damage in which exogenousdeoxyribonucleosides or derivatives thereof have useful therapeuticapplications.

Deoxyribonucleic acid has been used to accelerate wound cicatrization orhealing, and also to accelerate liver regeneration in experimentalanimals. It is likely that in these situations, as well as in thesituations where DNA is used to promote survival after irradiation ofanimals, the DNA is serving as a storage depot for deoxyribonucleosides,which gradually releases the deoxyribonucleotides anddeoxyribonucleosides during enzymatic degradation.

Administration of acylated deoxyribonucleosides, as described herein, isa method for delivering deoxyribonucleosides to tissues which ispreferable to the administration of foreign DNA for the purpose ofimproving wound healing or tissue regeneration. Unlike DNA, acylateddeoxyribonucleosides are effective after oral administration; they arealso nonantigenic and are much easier to purify than DNA.

The composition of the present invention may also be administered toenhance the healing of damaged tissue. Such damaged tissue includes skinwounds (e.g., punctures, lacerations, abrasions, etc.), burned tissue(skin, etc.), diseased or damaged liver (from surgery or other wounds ofthe liver, or from cirrhosis or diabetes, etc.), damaged heart muscle(e.g., improved scar formation after myocardial infarction), and damagedbone marrow (e.g., after radiation treatment or chemotherapeutictreatment).

For the purpose of treating skin wounds or burns, the compositions maybe applied topically as part of a skin lotion or cream, or as part of abioerodible polymer.

Preferred acyl substituent groups on the hydroxyl groups of thedeoxyribose ring of 2'-deoxyadenosine, 2'-deoxycytidine,2'-deoxyguanosine, and thymidine are fatty acids with 6 to 16 carbonatoms, or dicarboxylic acids with 4 to 6 carbon atoms, e.g., succinic,glutaric, or adipic acids. Preferred substituents on the exocyclic aminegroups of deoxycytidine, deoxyadenosine, and deoxyguanosine are aminoacids with basic side chains, e.g., lysine or arginine. Preferredsubstituents on the secondary amine in the thymine ring of thymidine arenicotinic acid or para-aminobenzoic acid.

Preferred deoxyadenosine derivatives comprise N⁶ -lysyl-5'-palmitoyldeoxyadenosine, 5'-palmitoyl adenosine, N⁶ -lysyl-5'-dodecanoyldeoxyadenosine, 5'-dodecanoyl adenosine, and N⁶ -lysyl-3',5'-diacetyldeoxyadenosine.

Preferred deoxyguanosine derivatives comprise N² -lysyl-5'-palmitoyldeoxyguanosine, 5'-dodecanoyl deoxyguanosine, and N²-lysyl-3',5'-diacetyl deoxyguanosine.

Preferred deoxycytidine derivatives comprise N⁴ -lysyl-5'-palmitoyldeoxycytidine, 5'-palmitoyl deoxycytidine, N⁴ -lysyl-5'-dodecanoyldeoxycytidine, 5'-dodecanoyl deoxycytidine, and N⁴ -lysyl-3',5'-acetyldeoxycytidine.

Preferred thymidine derivatives comprise N³ -nicotinoyl-5'-palmitoylthymidine, 5'-palmitoyl thymidine, N³ -nicotinoyl-5'-dodecanoylthymidine, 5'-dodecanoyl thymidine, and N³ -nicotinoyl-3',5'-acetylthymidine.

Compositions within the scope of the invention include those whichcontain mixtures of the acyl derivatives of the deoxyribonucleosides inamounts effective to achieve its intended purpose. Such compositions maycontain 0 to 50 mole percent of the acyl derivative of deoxycytidine, 0to 50 mole percent of the acyl derivative of deoxyguanosine, 0 to 50mole percent of the acyl derivative of deoxythymidine and 0 to 50 molepercent of the acyl derivative of deoxyadenosine, with the proviso thatthe total content of the acyl deoxyribonucleosides adds up to 100 molepercent.

A preferred composition contains 25 mole percent of the acyl derivativeof deoxycytidine, 25 mole percent of the acyl derivative ofdeoxyguanosine, 25 mole percent of the acyl derivative ofdeoxythymidine, and 25 mole percent of the acyl derivative ofdeoxyadenosine.

For treatment of radiation-induced cellular damage or sunburn, or toenhance wound healing, preferred dosages include amounts of the acylderivatives equivalent to 10 to 1000 mg of 2'-deoxyadenosine, 10 to 1000mg of 2'-deoxyguanosine, 10 to 1000 mg of 2'-deoxycytidine and 10 to1000 mg of 2'-deoxythymidine. For example, the composition may comprise13-1330 mg of 3',5'-diacetyl-2'-deoxyadenosine, 13-1310 mg of3',3'-diacetyl-2'-deoxyguanosine, 14-1370 mg of3',5'-diacetyl-2'-deoxycytidine and 14-1350 mg of3',5'-diacetyl-2'-deoxythymidine. As is understood in the art, incalculating such dosages, the equivalent amount of the2'-deoxyribnucleoside alone is considered, i.e., the acyl substituentand acid addition portion of any pharmaceutically acceptable salt arenot included in the calculation.

For a suntan lotion, 0.1 to 5% by weight of the above compositions maybe added. Generally, for this purpose, the acyl derivative will be inthe form of the free acyl deoxyribonucleosides and not as thepharmaceutically acceptable salts.

There are some situations in which it is useful to deliver a singledeoxyribonucleoside to tissues, e.g., deoxycytidine for treatment oftoxicity caused by the antineoplastic drug arabinosyl cytosine, orthymidine for treatment of toxicity caused by methotrexate. In suchcases, acyl derivatives of a single deoxyribonucleoside may beadministered.

Methods of Preparation

When the acid source of the desired acyl derivative has groups whichinterfere with the acylation reactions, e.g., hydroxyl or amino groups,these groups may be blocked with protecting groups, e.g.,t-butyldimethylsilyl ethers or t-BOC groups, respectively, beforepreparation of the anhydride. For example, lactic acid may be convertedto 2-t-butyldimethylsiloxypropionic acid witht-butyldimethylchlorosilane, followed by hydrolysis of the resultingsilyl ester with aqueous base. The anhydride may be formed by reactingthe protected acid with DCC. With-amino acids, the N-t-BOC derivativemay be prepared, using standard techniques, which is then converted tothe anhydride with DCC. With acids containing more than one carboxylategroup (e.g., succinic, fumaric, or adipic acid) the acid anhydride ofthe desired dicarboxylic acid is reacted with a 2'-deoxyribonucleosidein pyridine.

3',5'-Diacyldeoxythymidine may be prepared according to methodsdisclosed by Nishizawa et al., Biochem. Pharmacol. 14:1605 (1965), bytreating deoxythymidine with 2.1 equivalents of an acid anhydride of thedesired acyl compound in pyridine followed by heating to 80-85° C. forat least one hour. Alternatively, deoxythymidine may be treated with 2.1equivalents of an acid chloride in pyridine at room temperature. (SeeExample 1.)

The 5'-hydroxyl group of deoxythymidine may be selectively acylated with1 equivalent of the acid anhydride of the desired acyl compound inpyridine, which is heated to 80-85° C., according to Nishazawa, et al.Alternatively, the acid chloride (1 equivalent) may be reacted withdeoxythymidine in pyridine and DMF at room temperature according toBaker et al., J. Med. Chem. 21:1218 (1978). (See Example 2.)

The 3'-hydroxyl group of deoxythymidine may be selectively acylated byselectively forming the 5'-O-t-butyldimethylsilyl derivative with 1.2equivalents of t-butyldimethylchlorosilane in DMF containing imidizole,followed by acylation of the 3'-hydroxyl group with the appropriate acidanhydride, and cleavage of the 5'-t-butyldimethyl silyl ether accordingto Baker et al. (See Example 3.)

3',5'-Diacyldeoxycytidine may be prepared according to a method adaptedfrom Gish et al., J. Med. Chem. 14:1159 (1971), by treatingdeoxycytidine hydrochloride with 2.1 equivalents of the appropriate acidchloride in DMF. (See Example 5.)

The 5'-hydroxy group of deoxycytidine may be selectively acylated bytreating deoxycytidine hydrochloride with 1.1 equivalents of theappropriate acid anhydride in DMF. Gish et al. (See Example 6.)

The 3',5'-diacyl derivative of deoxyadenosine may be prepared bytreatment with 2.1 equivalents of the appropriate acid chloride in DMF.(Adapted from Gish et al., see Example 7.)

The 5'-hydroxyl group of deoxyadenosine may be selectively acylated bytreatment of deoxyadenosine hydrochloride with 1.1 equivalents of thedesired acid chloride in DMF. (Adapted from Gish et al., see Example 8.)

3',5'-Diacyl-2'-deoxyguanosine may be prepared by treatingdeoxyguanosine hydrochloride with 2.1 equivalents of the appropriateacid chloride in DMF. (Adapted from Gish et al., see Example 9.)

The 5'-hydroxyl group of deoxyguanosine may be selectively acylated bytreatment of deoxyguanosine hydrochloride with 1.1 equivalents of theappropriate acid chloride in DMF. (Adapted from Gish et al., see Example10.)

Amino acids may be coupled to the exocyclic amino groups ofdeoxyadenosine, deoxycytidine, and deoxyguanosine (or 3' or 5' acylderivatives thereof) by standard methods using dicyclohexylcarbodiimide.(See Example 11.)

These acyl compositions may be administered chronically to an animalwhich is at risk of either exposure to radiation, sunlight or chemicalmutagens. The acyl compositions of the invention may also beadministered after exposure to radiation, sunlight or chemical mutagensor after a wound is inflicted to enhance the repair of DNA and therebyto ameliorate the damage and promote survival of the animal.Advantageously, the compositions of the invention may be administeredbefore or after radiotherapy or chemotherapy to ameliorate undesiredside effects of the treatment.

The acyl compositions of the invention may also be coadministered withother radioprotective compounds such as WR-2721, NAC, DDC, cysteamine,2-mercaptoethanol, mercaptoethylamine, dithiothreitol, glutathione,2-mercaptoethanesulfonic acid, WR-1065, nicotinamide,5-hydroxytryptamine, 2-beta-aminoethyl-isothiouronium-Br-Hbr, glucans,GLP/BO4, GLP/BO5, OK-432, Biostim, PSK, Lentinan, Schizophyllan,Rhodexman, Levan, Mannozym, MVE-2, MNR, MMZ, IL-2, TNF, thymic factorTF-5, glutathione peroxidase, superoxide dismutase, catalase,glutathione reductase, glutathione transferase, selenium, CdCl₂, MnCl₂,Zn acetate, vitamin A, beta carotene, prostaglandins, tocopherol, andmethylene blue. The administration of these protective compounds alongwith the acyl derivatives of the invention provides protection greaterthan if the acyl derivatives or the other agents were given alone.

The pharmacologically active acyl derivatives may be combined withsuitable pharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds. Thesecan be administered as tablets, dragees, capsules, and suppositories.The compositions can be administered orally, rectally, vaginally, orreleased through the buccal pouch of the mouth, and may be applied insolution form by injection, orally or by topical administration. Thecompositions may contain from about 0.1 to 99%, preferably from about 50to 90% of the active compound(s), together with the excipient(s).

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compound(s) with solidexcipients, optionally grinding the resulting mixture and processing themixture of granules, after-adding suitable auxiliaries, if desired ornecessary, to obtain tablets or dragee cores.

Suitable excipients include fillers such as sugars, for example lactose,sucrose, mannitol or sorbitol, cellulose preparations and/or calciumphosphates, for example tricalcium phosphate or calcium hydrogenphosphate, as well as binders such as starch paste, using, for example,maize starch, wheat starch, rice starch or potato starch, gelatin,tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodiumcarboxymethyl cellulose, and/or polyvinyl pyrrolidone.

Auxiliaries are, above all, flow-regulating agents and lubricants, forexample, silica, talc, stearic acid or salts thereof, such as magnesiumstearate or calcium stearate, and/or polyethylene glycol. Dragee coresare provided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated sugar solutions may beused, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquersolutions and suitable organic solvents or solvent mixtures. In order toproduce coatings resistant to gastric juices, solutions of suitablecellulose preparations such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate are used. Dye stuffs or pigmentsmay be added to the tablets or dragee coatings, for example, foridentification or in order to characterize different combinations ofcompound doses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft-sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules contain the active compound(s) in the form of granules whichmay be mixed with fillers such as lactose, binders such as starchesand/or lubricants such as talc or magnesium stearate, and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids such as fatty oils, liquidparaffin, or polyethylene glycols. In addition, stabilizers may beadded.

Possible pharmaceutical preparations which can be used rectally include,for example, suppositories which consist of a combination of activecompounds with a suppository base. Suitable suppository bases are, forexample, natural or synthetic triglycerides, paraffin hydrocarbons,polyethylene glycols or higher alkanols. In addition, it is alsopossible to use gelatin rectal capsules which consist of a combinationof the active compounds with a base. Possible base materials include,for example, liquid triglycerides, polyethylene glycols, or paraffinhydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water soluble form, for example,water soluble salts. In addition, suspensions of the active compounds asappropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may include substanceswhich increase the viscosity of the suspension which include, forexample, sodium carboxymethylcellulose, sorbitol, and/or dextran.Optionally, the suspension may also contain stabilizers.

The acyl deoxyribonucleosides may be formulated as part of a skin lotionor suntan lotion for topical administration. Suitable formulations fortopical administration include appropriate oily suspensions orsolutions. Suitable lipophilic solvents or vehicles include fatty oils,for example sesame oil or coconut oil, or synthetic fatty acid esters,for example ethyl oleate or triglycerides. These topical formulationsmay be used to treat damaged tissue such as skin wounds or burns, or totreat or prevent sunlight induced cellular damage (sunburn).

For purposes of enhancing wound healing, the compositions of the presentinvention may be formulated as part of wound dressings, or incorporatedinto bioerodible microcapsules-for topical administration. Suchmicrocapsules may comprise, for example, polylactate orlactate-glycolate copolymers. See Weise, D. L. et al., Drug Carriers inBiology and Medicine, Gregoriadis, G. et al., Academic Press, N.Y. p.237-270 (1979).

The following examples are illustrative, but not limiting of the methodsand compositions of the present invention. Other suitable modificationsand adaptations of a variety of conditions and parameters normallyencountered in clinical therapy which are obvious to the those skilledin the art are within the spirit and scope of this invention.

EXAMPLES OF METHODS TO PREPARE COMPOUNDS OF THE INVENTION Example 1Preparation of 3',5'-Diacyl-2'-deoxythymidine From Acid Anhydrides

2'-Deoxythymidine is dissolved in anhydrous pyridine at roomtemperature. 2.1 molar equivalents of the acid anhydride of the desiredacyl compound (e.g., acetic anhydride, lactate anhydride, butyricanhydride, etc.) is then added. The reaction mixture is then heated to80-85° C. for 1 to 4 hours, cooled, poured into ice water, and theesters recovered by extraction with chloroform or a similar solvent. Thechloroform is then washed with ice-cold 0.01 N sulfuric acid, 1% aqueoussodium bicarbonate, and finally water. After drying with sodium sulfate,the chloroform is evaporated and the residual oil or crystals aresubjected to chromatography (adapted from Nishizawa et al., Biochem.Pharmacol. 14:1605 (1965)).

From Acid Chlorides

To 2'-deoxythymidine dissolved in anhydrous pyridine is added, at 5° C.,2.1 molar equivalents of the acid chloride of the desired acyl compound(e.g., palmitoyl chloride, acetyl chloride, etc.). The mixture is heldat room temperature overnight, added to ice water, and worked up asindicated above (adapted from Nishizawa).

Example 2 Preparation of 5'-Acyl-2'-deoxythymidine

To 2'-deoxythymidine dissolved in anhydrous pyridine is added, at roomtemperature, 1.0 molar equivalent of the acid anhydride of the desiredacyl compound. The reaction is then heated to approximately 80-85° C.for several hours, cooled, poured into ice water, and the estersrecovered by extraction with chloroform or a similar solvent. Thechloroform is then washed in ice-cold 0.01 N sulfuric acid, 1% aqueoussodium bicarbonate, and finally water. After drying with sodium sulfate,the chloroform is evaporated and the residual oil or crystals aresubjected to chromatography. The major product, which is isolated bychromatography is the 5' substituted ester (adapted from Nishizawa etal.)

Alternatively, selectively 5' acylation of deoxythymidine may beaccomplished by suspending 2'-deoxythymidine in a mixture of pyridineand N,N-dimethylformamide cooled to 0° C. in an ice bath. 1.0 molarequivalent of the acid chloride of the desired acyl compound is addeddropwise to the mixture, which is stirred at 9° C. for 12-24 hours.Water is then added to stop the reaction, and then the solvents areevaporated in vacuo at 50° C. The residue is dissolved in methanol andpurified by chromatography on silica gel (adapted from Baker et al., J.Med. Chem. 21:1218 (1978)).

Example 3 Preparation of 3'-Acyl-2'-deoxythymidine

To a stirred suspension of 2'-deoxythymidine in dryN,N-dimethylformamide is added 2.4 molar equivalents of imidazolefollowed by 1.2 molar equivalents of t-butyldimethylchlorosilane. Themixture is stirred with protection from moisture at room temperature for20 hours, at which time the solvent is removed at 50° C. in vacuo. Theresidue is dissolved in 15 ml of ethyl acetate, washed, and evaporatedto give a syrup from which is obtained, by crystallization from hotchloroform by the addition of hexane to the point of opalescence,5'-(t-butyldimethylsilyl)-2'-deoxythymidine.

To a stirred suspension of 5'-(t-butylmethylsilyl)-2'-deoxythymidine indry pyridine cooled to 0° C. is added 1.1 molar equivalents of theappropriate acid anhydride of the desired acyl compound, and the mixtureis stirred with protection from moisture for 20 hours at 0-5° C., atwhich time the reaction is terminated by addition of a few ml of water.The solvent is evaporated and the residue is extracted and evaporated togive a thick, clear syrup, which is then dried in vacuo at 25° C.

The t-butylmethylsilyl group is removed with glacial acetic acid andtetrabutylammonium fluoride in tetrahydrofuran, yielding the desired3'-acyl-2'-deoxythymidine derivative (adapted from Baker et al.).

Example 4 Preparation of N³ -Acyl-2'-deoxythymidine

The acylation of the secondary amine in the 3 position of the pyrimidinering is accomplished by reacting 3',5'-diacyldeoxythymidine with 1.1molar equivalents of the acid chloride of the desired acyl substituentin an aprotic solvent (such as ether, dioxane, chloroform, ethylacetate, acetonitrile, pyridine, dimethylformamide, and the like) in thepresence of 1-5 molar equivalents of an organic base (especiallyaromatic amines such as pyridine, trialkylamines, orN,N-dialkylanilines) (adapted from Fuji et al., U.S. Pat. No.4,425,335). The acyl substituent on the secondary amine can be the sameor different from those on the hydroxyl groups of the ribose moiety.

Example 5 Preparation of 3',5'-Diacyl-2'-deoxycytidine

2-Deoxycytidine hydrochloride is dissolved in N,N-dimethylformamide. 2.1molar equivalents of the acid chloride of the desired acyl substituentis added, and the mixture is stirred overnight at room temperature. Thereaction mixture is concentrated in vacuo to an oil, and triturated witha mixture of ethyl acetate and diethyl ether or similar solvents. Theoil is then triturated with IN sodium hydrogen carbonate. Thecrystalline solid is collected, washed with water, dried, andrecrystallized (adapted from Gish et al., J. Med. Chem. 14:1159 (1971)).

Example 6 Preparation of 5'-Acyl-2'-deoxycytidine

2-Deoxycytidine hydrochloride is dissolved in N,N-dimethylformamide. 1.1molar equivalents of the acid chloride of the desired acyl substituentis added, and the mixture is stirred overnight at room temperature. Thereaction mixture is concentrated in vacuo to an oil, and triturated witha mixture of ethyl acetate and diethyl ether or similar solvents. Theoil is then triturated with 1N sodium hydrogen carbonate. Thecrystalline solid is collected, washed with water, dried, andrecrystallized (adapted from Gish et al.).

Example 7 Preparation of 3',5'-Diacyl-2'-deoxyadenosine

2'-Deoxyadenosine is dissolved in N,N-dimethylformamide and pyridine(1:1). 2.1 molar equivalents of the acid chloride of the desired acylsubstituent is added, and the mixture is stirred overnight at roomtemperature. The reaction mixture is concentrated in vacuo to an oil,and triturated with a mixture of ethyl acetate and diethyl ether orsimilar solvents. The oil is then triturated with 1N sodium hydrogencarbonate. The crystalline solid is collected, washed with water, dried,and recrystallized.

Example 8 Preparation of 5'-Acyl-2'-deoxyadenosine

2'-Deoxyadenosine is dissolved in N,N-dimethylformamide and pyridine(1:1). 1.1 molar equivalents of the acid chloride of the desired acylsubstituent is added, and the mixture is stirred overnight at roomtemperature. The reaction mixture is concentrated in vacuo to an oil,and triturated with a mixture of ethyl acetate and diethyl ether orsimilar solvents. The oil is then triturated with 1N sodium hydrogencarbonate. The crystalline solid is collected, washed with water, dried,and recrystallized.

Example 9 Preparation of 3',5'-Diacyl-2'-deoxyguanosine

2'-Deoxyguanosine is dissolved in N,N-dimethylformamide and pyridine(1:1). 2.1 molar equivalents of the acid chloride of the desired acylsubstituent is added, and the mixture is stirred overnight at roomtemperature. The reaction mixture is concentrated in vacuo to an oil,and triturated with a mixture of ethyl acetate and diethyl ether orsimilar solvents. The oil is then triturated with 1N sodium hydrogencarbonate. The crystalline solid is collected, washed with water, dried,and recrystallized.

Example 10 Preparation of 5'-Acyl-2'-deoxyguanosine

2'-Deoxyguanosine is dissolved in N,N-dimethylformamide and pyridine.1.1 molar equivalents of the acid chloride of the desired acylsubstituent is added, and the mixture is stirred overnight at roomtemperature. The reaction mixture is concentrated in vacuo to an oil,and triturated with a mixture of ethyl acetate and diethyl ether orsimilar solvents. The oil is then triturated with 1N sodium hydrogencarbonate. The crystalline solid is collected, washed with water, dried,and recrystallized (adapted from Gish et al.).

Example 11 Synthesis of N-lysyl-5'-O-palmitoyl-deoxycytidine

5'-O-palmitoyldeoxycytidine is synthesized by reacting deoxycytidinehydrochloride with 1.1 equivalents palmitoyl chloride in drydimethylformamide.

14 grams of 5'-O-palmitoyl-deoxycytidine is dissolved in 100 mldimethylacetamide, 1 molar equivalent of di-tert-butoxycarbonyl-lysineis added, and the mixture is cooled in an ice bath. 1.2 molarequivalents (7.4 g) of dicyclohexylcarbodiimide are added and themixture is stirred at 4° C. for 90 hours. The precipitate(dicyclohexylurea) is removed by filtration. 100 ml of water is added tothe filtrate, followed by 1 liter of ethyl acetate. N⁴-(di-N-tert-butoxycarbonyl-lysyl)-5'-O-palmitoyl-deoxycyt-idine isseparated from unreacted reagents by chromatography over silica gel. Thet-butoxycarbonyl protecting groups are removed by the standard methodsof treatment with an acid, such as trifluoroacetic acid.

Similarly, 5'-O-acyl or 3',5'-O-diacyl derivatives of deoxycytidine,deoxyadenosine, or deoxyguanosine in general may have lysine or argininecoupled to their exocyclic primary amino groups withdicyclohexylcarbodiimide.

Examples of Protection of Nucleosides from Enzymatic Degradation byAcylation

Deoxyribonucleosides are rapidly degraded following administration toanimals. In order to successfully utilize acylated nucleosides todeliver nucleosides to tissues, it is imperative that acylation shouldprevent degradation of the nucleoside moiety by the enzymes thatnormally degrade the nucleosides. For each of the majordeoxyribonucleosides, a different enzyme is involved in the first stepof their degradation. The first step in degradation of deoxyadenosine isdeamination catalyzed by adenosine deaminase. Thymidine is initallycatabolized by thymidine phosphorylase; deoxyguanosine by purinenucleoside phyosphorylase, and deoxycytidine is degraded bydeoxycytidine deaminase.

Example 17

Solutions (100 micromolar in phosphate-buffeed saline) of each of thedeoxyribonucleosides or their acylated derivatives were incubated at 37°C. with each of the four enzymes: adenosine deaminase (ADA), cytidinedeaminase (CDA), purine nucleoside phosphorylase (PNP), and thymidinephosphorylase (TP). Enzymatic degradation of compounds was determined byHPLC.

                  TABLE 1                                                         ______________________________________                                        Protection of Deoxyribonucleosides                                            From Enzymes Which Catalyze                                                   Initial Steps of Degradation                                                  By 5'-0-Acylation                                                                              Enzyme                                                       Compound          ADA      CDA    PNP    TP                                   ______________________________________                                        deoxyadenosine    +        -      -      -                                    3',5'-di-O-acetyldeoxyadenosine                                                                 -        -      -      -                                    5',-O-palmitoyldeoxyadenosine                                                                   -        -      -      -                                    deoxycytidine     -        +      -      -                                    3',5'-di-O-acetyldeoxycytidine                                                                  -        -      -      -                                    5',-O-palmitoyldeoxycytidine                                                                    -        -      -      -                                    deoxyguanosine    -        -      +      -                                    3',5'-di-O-acetyldeoxyguanosine                                                                 -        -      -      -                                    5',-O-palmitoyldeoxyguanosine                                                                   -        -      -      -                                    thymidine         -        -      -      +                                    3',5'-di-O-acetylthymidine                                                                      -        -      -      -                                    5'-O-valerylthymidine                                                                           -        -      -      -                                    5'-O-octanoylthymidine                                                                          -        -      -      -                                    5'-O-palmitoylthymidine                                                                         -        -      -      -                                    5'-O-valerylthymidine                                                                           -        -      -      -                                    ______________________________________                                         + indicates that compound was a substrate for the enzyme.                     - indicates that compound was not a substrate for the enzyme.            

These data show that 5'-O-acylation of deoxyribonucleosides protectsthem from the enzymes that catalyze the initial steps of theirdegradation. Thus, the nucleoside moiety of acylated nucleosides willremain intact in vivo until deacylation occurs.

Examples of Deacylation of Acylated Deoxyribonucleosides in LiverExtracts

Acylated deoxyribonucleosides were incubated with rat liver extracts inorder to assess the relative rates of enzymatic deacylation ofderivatives with different substituents, and to determine whetherdifferent nucleosides with the same substituents are deacylated atsimilar rates. Deacylation of the derivatives of the invention in vivoresults in release of the parent nucleosides, which can then be utilizedby cells.

Example 12

Whole rat livers were homogenized in phosphate-buffered saline (10 mlper gram of liver) and centrifuged. The supernatant was diluted to afinal concentration of 50 ml buffer per gram of liver, and stocksolutions of acylated deoxyribonucleosides were added so that thecompounds were present at concentrations of 100 micromolar. 100microliter aliquots were removed periodically to determine, by HPLC, theamounts of free nucleosides produced as a function of time.

                  TABLE 2                                                         ______________________________________                                                      Nucleosides Released (nanomoles/ml)                                           Hours                                                           Compound        1       2      3     8    24                                  ______________________________________                                        3',5'-di-O-acetyldeoxyadenosine                                                               26      39     57                                             3',5'-di-O-acetyldeoxycytidine                                                                25      40     50                                             3',5'-di-O-acetyldeoxyquanosine                                                               23      43     63                                             3',5'-di-O-acetylthymidine                                                                    19      40     63                                             5'-O-valerylthymidine                                                                         47      95     98                                             5'-O-octanoylthymidine                                                                        74      84     96                                             5'-O-acetyldeoxyadenosine                                                                     48                                                            5'-O-valeryldeoxyadenosine                                                                    65                                                            N-acetyldeoxycytidine                                                                          0       0      0    0                                        N-valeryldeoxyguanosine                                                                        0       0      0    0                                        N-palmitoyldeoxyguanosine                                                                      0       0      0    0                                        5'-O-palmitoyldeoxyadenosine         5    16                                  5'-O-palmitoyldeoxycytidine          4    15                                  5'-O-palmitoyldeoxyguanosine         3    14                                  5'-O-palmitoylthymidine              4    14                                  ______________________________________                                    

These data indicate that in liver extracts, di-O-acetyl derivatives ofeach of the four deoxyribonucleosides are deacylated at very similarrates. This is also true for 5'-O-palmitoyl derivatives, although thepalmitoyl substituents are cleaved at a much slower rate than are theacetate groups. This suggests that the rate of deacylation ofO-substituted deoxyribonucleosides is primarily a function of the natureof the acyl substituent, and not the nucleoside to which it is attached.This is important in the practice of the invention, since sometherapeutic effects are obtained only when derivatives of more than onenucleoside are coadministered. It is preferable if deacylation ofdifferent nucleosides in a therapeutic mixture occurs at similar ratesin vivo because optimal proportions of nucleosides are delivered totissues simultaneously. The large variation in deacylation rates fornucleosides substituted with short chain (acetyl) versus long chain(palmitoyl) fatty acids gives rise to the opportunity for selecting acylsubstituents according to the rates of deacylation (or rates ofnucleoside delivery) required in different clinical situations.Midlength fatty acid substituents (e.g., valeryl and octanoyl) arecleaved more rapidly than are shorter (acetate) or longer (palmitate)substituents.

In this same liver extract, deoxyribonucleosides per se are rapidlydegraded, e.g., deoxyadenosine at a concentration of 100 micromolar isentirely degraded, initially by deamination to form inosine, within 2minutes. Thus, it can be understood that following administration, theO-acylated deoxyribonucleosides of this invention will graduallyrelease, and provide to tissues, free nucleosides for a sustained periodof time, compared to the brief period of nucleoside availabilityfollowing administration of the parent deoxyribonucleosides themselves.Fatty acids on the primary amines of either the pyrimidine ring ofdeoxycytidine or the purine rings of deoxyguanosine are not removed atan appreciable rate by liver enzymes.

Examples of Oral Administration of Acylated Nucleosides

In order to demonstrate delivery of deoxyribonucleosides after oraladministration of acylated nucleosides, plasma thymidine levels weremeasured after oral administration of either 3',5'-di-O-acetylthymidineor thymidine itself.

Example 13

Male F344 rats (350 g) were implanted with chronic jugular veincatheters for blood sampling and allowed to recover for two days. Abasal blood sample was taken, and then 0.7 millimoles of thymidine or3',5'-di-O-acetylthymidine (DAT) were administered by gavage. Bloodsamples were withdrawn at 0.5, 1, 2, and 4 hours after administration,centrifuged, and the supernatant (plasma) was deproteinized withmethanol. The concentration of thymidine in the plasma samples wasdetermined by HPLC with UV absorbance detection.

The basal plasma thymidine concentration was 1 micromolar. Followingoral administration of thymidine, plasma thymidine levels reached amaximum of 9 micromolar one hour after administration and returned tobasal concentrations by 4 hours. In contrast, following oraladministration of an equimolar dose of 3',5'-di-O-acetylthymidine,plasma thymidine concentrations reached a maximum of 80 micromolarwithin 30 minutes, and were still elevated above basal values four hoursafter administration.

Thus, oral administration of di-O-acetylthymidine delivers much greaterquantities of thymidine to tissues (over a longer duration) than doesadministration of the nonderivatized nucleoside as is shown in FIG. 4wherein the comparative data are plotted.

Examples of Clinical Administration Radiation Exposure

Three situations wherein acyl derivatives of deoxyribonucleosides may beclinically useful in treating radiation damage are 1) accidentalexposure to ionizing radiation, as in a nuclear accident; 2) exposure toX-radiation during radiography; and 3) radiotherapy of cancer.

In the first case, acyl deoxyribonucleoside derivatives should beadministered in a formulation suitable for parenteral injection,followed by oral administration several times per day of dosesequivalent to 0.5 to 2 grams of each of the four majordeoxyribonucleosides. It is essential that the derivatives of all of thenucleosides be coadministered.

In the second case, X-ray exposure during diagnostic radiography, acyldeoxyribonucleside derivatives are given orally before and afterexposure.

In the third case, during cancer radiotherapy, the acyl ribonucleosidederivatives are particularly useful in restoring bone marrow functionafter its undesirable but unavoidable suppression during irradiation.Moreover, in formulations designed to selectively deliver nucleosides tonormal but not neoplastic tissues, the acyl nucleoside derivatives willimprove the therapeutic index (ratio of efficacy to toxicity) of theradiation treatment. Similar doses of deoxyribonucleoside derivativesmay also be used to treat bone marrow suppression caused byantineoplastic or antiviral chemotherapy.

The following example discloses the benefits of the invention in thetreatment of irradiated mice.

Methods

Balb/c+ mice were subjected to gamma irradiation (Cobalt 60) at a doserate of 7.3 Rads/min. The field was measured twice by Fricke dosimetryto ensure field uniformity and dose constancy for each mouse. Groups of15 mice received total doses of gamma radiation of 675, 700, 725, and750 R.

Mice were divided into four treatment groups (5 mice at each radiationdose), each receiving a different post-irradiation treatment:

Group 1: 0.9% saline (control group);

Group 2: A mixture of deoxyribonucleosides (equimolar mixture ofdeoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine);

Group 3: A mixture of the 3,5'-di-O-palmitoyl derivatives ofdeoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine, equimolarto the doses of non-derivatized nucleosides.

Group 4: A mixture of the 5'-O-acetyl derivatives of deoxyadenosine,deoxyguanosine, deoxycytidine, and thymidine. (Tested at 750 R only.)

The nucleosides or di-O-acetyl derivatives were administered byintraperitoneal injection (8 micromoles/0.2 ml physiological salinethree times daily (every 8 hours) for 4 days, beginning 30 minutes afterirradiation). Mice in the control groups received injections of 0.2 mlphysiological saline on the same schedule. Mice receiving 5'-O-palmitoylnucleoside derivatives were given 8 micromoles only once per day for the4 days following irradiation; thus, they received only one third of themolar quantity of nucleosides given to the mice receiving eithernonderivatized or acetylated nucleosides.

Mortality was monitored daily for 30 days.

Results and Discussion

The LD 50/30 (the radiation dose that produces 50% mortality within 30days after irradiation) in this strain of mice is approximately 650 R.At the radiation doses tested, death occurred, if at all, 12 to 20 daysafter irradiation, which is characteristic of lethal post-irradiationbone-marrow failure.

At the lowest radiation dose tested in this experiment (675 R), only 20%of the saline-treated control mice survived; no control animals survivedat any of the higher radiation doses (Table 1). Post-irradiationadministration of deoxyribonucleosides did not significantly improvesurvival over that of mice in the control group. In contrast, animalstreated with the di-O-acetyl deoxyribonucleosides after irradiationsurvived radiation doses which were lethal to animals given eitherdeoxyribonucleosides or saline (700 R through 750 R). Mice treated with5'-O-palmitoyl deoxyribonucleosides also survived radiation doses thatwere lethal to untreated mice (750 R). It is apparent that the palmitoylderivatives (8 micromoles administered once per day) are at least aseffective in improving survival of irradiated mice as a threefold higherdose of di-O-acetyl nucleosides (8 micromoles administered three timesper day).

Agents that improve survival when administered after irradiation do soby improving proliferation and differentiation of survivinghematopoietic stem cells. It is therefore apparent that the nucleosidederivatives of the invention will be useful in other situations of bonemarrow impairment, such as occurs after treatment with certainantineoplastic agents.

                  TABLE 3                                                         ______________________________________                                        Percentage of mice surviving                                                  at 30 days after potentially                                                  lethal gamma irradiation                                                                     % Survival at 30 Days                                                         Radiation Dose (R)                                             Treatment        675     700     725   750                                    ______________________________________                                        saline (control)  20      0       0     0                                     deoxyribonucleosides                                                                            40      0       0     0                                     di-O-acetyldeoxyribonucleosides                                                                100     100     100    80                                    5'-O-palmitoyl-                                                                                100                                                          deoxyribonucleosides                                                          ______________________________________                                         Mice were treated with the listed agents after gamma irradiation as           described in the text.                                                   

Wound Healing

In promoting the healing of skin wounds (whether surgical incisions oraccidental wounds), it is best to apply acyl deoxyribonucleosidederivatives topically, either in an ointment, in bioerodiblemicrocapsules, or incorporated into wound dressings. A topicalantibiotic might be coadministered. The molar equivalent of 2 to 20 mgof a mixture of all four major deoxyribonucleosides should be appliedper square cm of wound area, or 1 to 10 mg per cm of linear incision.The onset of the earliest phases of wound healing in particular isaccelerated.

Liver Regeneration

Acyl derivatives of deoxyribonucleosides are useful in promotingregeneration of damaged or diseased liver, particularly for acceleratingregrowth after surgical removal of a portion of the liver. In this case,oral administration of the derivatives is preferable, in dosescorresponding to the molar equivalents of 0.2 to 2 grams of eachnucleoside. It is important that derivatives of all four majordeoxyribonucleosides be coadministered.

What is claimed is:
 1. A method of enhancing hematopoiesis in an animalin need of such treatment comprising administering to said animal ahematopoiesis stimulating amount of one or more compounds selected fromthe group consisting of ##STR19## wherein R₁, R₂, and R₃ are the same ordifferent and each is hydrogen or an acyl group derived from(a) anunbranched fatty acid with 2 to 22 carbon atoms, (b) an amino acidselected from the group consisting of glycine, the L forms of alanine,valine, leucine, isoleucine, tyrosine, proline, hydroxyproline, serine,threonine, cysteine, aspartic acid, glutamic acid, arginine, lysine,histidine, carnitine, and ornithine, (c) nicotinic acid, or (d) adicarboxylic acid having 3 to 22 carbon atoms,provided that not all ofR₁, R₂, and R₃ are H, or a pharmaceutically acceptable salt thereof. 2.A method as in claim 1 wherein said compound is ##STR20## wherein R₁,R₂, and R₃ are the same or different and each is hydrogen or an acylgroup derived from(a) an unbranched fatty acid with 2 to 22 carbonatoms, (b) an amino acid selected from the group consisting of glycine,the L forms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cysteine, aspartic acid, glutamicacid, arginine, lysine, histidine, carnitine, and ornithine, (c)nicotinic acid, or (d) a dicarboxylic acid having 3 to 22 carbonatoms,provided that not all of R₁, R₂, and R₃ are H, or apharmaceutically acceptable salt thereof.
 3. A method as in claim 1wherein said compound is ##STR21## wherein R₁, R₂, and R₃ are the sameor different and each is hydrogen or an acyl group derived from(a) anunbranched fatty acid with 2 to 22 carbon atoms, (b) an amino acidselected from the group consisting of glycine, the L forms of alanine,valine, leucine, isoleucine, tyrosine, proline, hydroxyproline, serine,threonine, cysteine, aspartic acid, glutamic acid, arginine, lysine,histidine, camitine, and ornithine, (c) nicotinic acid, or (d) adicarboxylic acid having 3 to 22 carbon atoms,provided that not all ofR₁, R₂, and R₃ are H, or a pharmaceutically acceptable salt thereof. 4.A method as in claim 1 wherein said compound is ##STR22## wherein R₁,R₂, and R₃ are the same or different and each is hydrogen or an acylgroup derived from(a) an unbranched fatty acid with 2 to 22 carbonatoms, (b) an amino acid selected from the group consisting of glycine,the L forms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyprolne, serine, threonine, cysteine, aspartic acid, glutamicacid, arginine, lysine, histidine, carnitine, and ornithine, (c)nicotinic acid, or (d) a dicarboxylic acid having 3 to 22 carbonatoms,provided that not all of R₁, R₂, and R₃ are H, or apharmaceutically acceptable salt thereof.
 5. A method as in claim 1wherein said compound is ##STR23## wherein R₁, R₂, and R₃ are the sameor different and each is hydrogen or an acyl group derived from(a) anunbranched fatty acid with 2 to 22 carbon atoms, (b) an amino acidselected from the group consisting of glycine, the L forms of alanine,valine, leucine, isoleucine, tyrosine, proline, hydroxyproline, serine,threonine, cysteine, aspartic acid, glutamic acid, arginine, lysine,histidine, carnitine, and ornithine, (c) nicotinic acid, or (d) adicarboxylic acid having 3 to 22 carbon atoms,provided that not all ofR₁, R₂, and R₃ are H, or a pharmaceutically acceptable salt thereof. 6.A method as in claim 2 wherein R₁, R₂ and R₃ are the same or differentand each is an unbranched fatty acid with 3-22 carbon atoms.
 7. A methodas in claim 3 wherein R₁, R₂ and R₃ are the same or different and eachis an unbranched fatty acid with 3-22 carbon atoms.
 8. A method as inclaim 4 wherein R₁, R₂ and R₃ are the same or different and each is anunbranched fatty acid with 3-22 carbon atoms.
 9. A method as in claim 5wherein R₁, R₂ and R₃ are the same or different and each is anunbranched fatty acid with 3-22 carbon atoms.